Delayed cerebral ischaemia (DCI) is a major determinant of poor clinical outcome after aneurysmal subarachnoid haemorrhage (aSAH), but its underlying mechanisms are not fully understood. We aimed to perform a genome-wide association study (GWAS) in aSAH patients with and without DCI. We conducted a multicentre study of European ancestry within the International Stroke Genetics Consortium with two stages: Stage 1 (n = 1,085; four centres) and Stage 2 (n = 801; one centre). A GWAS meta-analysis was performed across both stages. We then examined whether variants associated with DCI were linked to differences in gene expression (expression quantitative trait loci, eQTLs) or DNA methylation (methylation quantitative trait loci, mQTLs) and validated the methylation findings in an aSAH cohort with available DNA methylation data (n = 344). We performed complementary postGWAS analysis in order to promote biological interpretation of the genome-wide findings, including the genetic overlap with ischaemic stroke and intracranial aneurysm (ruptured and unruptured) using publicly available published GWAS summary statistics. Among 1,879 patients with DCI data, the prevalence of DCI was 22.8% (428/1,879). In the meta-analysis across both stages, we identified one genome-wide significant variant, rs7636350 (Chr3:130328911:T:C), associated with an increased risk of DCI (additive OR 1.75 [95% CI 1.43-2.13], P=4.47×10⁻⁸). This variant mapped near the COL6A6 and PIK3R4 genes. Functional annotation revealed one significant eQTL for PIK3R4 and one mQTL for cg23181900, located within COL6A6. Hypermethylation at cg23181900 was associated with DCI in a cohort with DNA methylation data (logFC=0.016, 95% CI 0.002-0.032; P=0.0317). Genetic correlation analyses showed a marginal correlation with ischaemic stroke due to large artery atherosclerosis (r=0.172; P=0.0508) and a higher correlation with intracranial aneurysm (r=0.341; P=0.002). We identified a genome-wide significant locus associated with DCI after aSAH, implicating COL6A6 and PIK3R4. We additionally identified an mQTL at cg23181900, suggesting a regulatory link at this locus. These findings enhance our understanding of DCI pathophysiology and support further studies to clarify causality and determine whether these pathways may inform future therapeutic strategies.

Brain arteriovenous malformations (bAVMs) are a major cause of intracranial haemorrhage in children and young adults, yet the molecular basis of endothelial dysfunction and vascular fragility remains incompletely understood. Although somatic activation of the KRAS-MAPK pathway has been implicated in bAVM pathogenesis, the contribution of epigenetic dysregulation has not been systematically defined. We performed genome-wide DNA methylation profiling using the Illumina Infinium MethylationEPIC v2 (935K) array on human bAVM nidus tissues, followed by targeted pyrosequencing and immunofluorescence validation. Functional and mechanistic studies were conducted in human brain microvascular endothelial cells, complemented by transcriptomic analyses and in vivo assessment in an endothelial-specific zebrafish model. Genome-wide methylation analysis revealed a distinct promoter-centric epigenetic landscape in bAVM tissue, with enrichment of genes involved in cell adhesion, angiogenesis and inflammatory signalling. Among these, Nerve Injury-Induced Protein 1 (NINJ1) emerged as a prominently hypomethylated gene with increased endothelial expression in bAVM lesions. In human brain microvascular endothelial cells, NINJ1 overexpression enhanced proliferation, migration and tube formation, whereas NINJ1 knockdown produced opposite effects. DNMT1 silencing upregulated NINJ1 expression and induced angiogenic behaviour, which was abolished by concurrent NINJ1 knockdown. Mechanistically, NINJ1 enhanced KRAS-MAPK/ERK signalling, at least in part by increasing KRAS protein stability. NINJ1 overexpression also increased endothelial permeability and reduced VE-cadherin and ZO-1 expression. In vivo, endothelial-specific overexpression of ninj1 in zebrafish impaired vascular integrity and induced intracranial haemorrhage. These findings identify promoter hypomethylation-associated activation of NINJ1 as a previously unrecognised epigenetic contributor to endothelial dysfunction in bAVM and support a role for NINJ1 as an upstream enhancer of KRAS-MAPK signalling. Epigenetic dysregulation of NINJ1 may therefore contribute to vascular instability and haemorrhage risk in bAVM.

SORL1, the gene encoding the SORLA protein, has arisen as a potential therapeutic target for Alzheimer's disease (AD). Studies suggest that restoring SORLA function or its trafficking pathways, particularly the SORLA-retromer recycling system, may offer a promising strategy to slow or halt AD progression. While both rare and common SORL1 variants have been associated with increased AD risk, recent evidence suggests a potential involvement of SORL1 in other neurodegenerative conditions. This study assessed the contribution of SORL1 genetic variation to the risk of AD, related dementias (RD), and Parkinson's disease (PD) using data from six large-scale biobanks, comprising 15,043 AD, 9,943 RD, and 42,763 PD cases, along with 111,969 controls across 11 ancestries. We identified 53 potentially disease-related SORL1 variants (CADD score > 20, MAC ≥ 2, annotated as protein-altering or splicing, and with the mutated allele present only in cases), including 41 novel and 12 previously reported variants. Three were found across multiple ancestries. Overall, 13 variants were found in AD-related cohorts, 5 in RD cohorts, and 35 in PD cohorts. Association analysis identified 10 nominally significant variants associated with AD and 5 with PD. The replication of multiple SORL1 variants across neurodegenerative diseases and ancestrally diverse populations underscores its potential broad genetic contribution to neurodegeneration and reinforces its relevance across distinct clinical phenotypes. Burden analysis identified a nominal association of SORL1 variants in PD in the South Asian population (P = 0.048). A family-based analysis identified a rare predicted-damaging variant in two East Asian families (11:121478242:G:A, p.R176Q) and two variants in two families of European ancestry (11:121514222:A:C, p.N371T; 11:121545392:G:A, p.V672M) that show some evidence of segregation in PD families. Although these variants were slightly more frequent in unrelated PD cases vs. controls, none of them showed statistically significant enrichment in PD, likely due to their very low frequency. Overall, our results extend the understanding of SORL1 beyond AD, suggesting a broader role in neurodegeneration and emphasizing the need for diverse population studies when evaluating genetic risk.

Recent advances in anti-amyloid therapies for Alzheimer's disease have been promising, but they have also highlighted critical challenges, including increased vascular complications, such as amyloid-related imaging abnormalities. Emerging evidence suggests that the soluble epoxide hydrolase may be a promising therapeutic target due to the involvement of sEH-derived diols in inflammation, oxidative stress, and vascular destabilization. APPPS1 mice, a model of amyloidosis, were crossed with an inducible soluble epoxide hydrolase knock-out mouse line. The knock-out was induced before onset of amyloid deposition, and then the mice were analyzed using histological, molecular, and RNA sequencing techniques. Here, we identify astrocytic soluble epoxide hydrolase as a key mediator of vascular instability in amyloid pathology. Targeted astrocyte-specific deletion of soluble epoxide hydrolase in APPPS1 mice dramatically mitigated vascular changes, reducing the vascular amyloid burden by 67.95% and preserving VE-cadherin architecture. Importantly, vasomotion was markedly impaired in the Alzheimer's disease model and was preserved in soluble epoxide hydrolase-deficient animals. Transcriptomic profiling of vasculature in APPPS1xsEHΔAC mice revealed upregulated expression of genes critical for neurovascular protection. These findings identify soluble epoxide hydrolase as a central regulator of neurovascular dysfunction and underscore its therapeutic potential in increasing vascular stability in amyloidosis-associated diseases, such as Alzheimer's disease.

Resting tremor in Parkinson's disease is characterized by considerable variability: tremor affects some patients but not others, and its amplitude typically fluctuates. Previous research focused primarily on identifying mechanisms that produce tremor, showing an important role for the basal ganglia and cerebello-thalamo-cortical circuit. What has been relatively ignored are mechanisms involved in suppressing tremor. For instance, patients can reduce their tremor by making voluntary movements or by exerting conscious effort. Here, we aimed to distinguish between brain mechanisms that contribute positively or negatively to spontaneous fluctuations in Parkinson's tremor. We leveraged a large sample of 119 tremulous Parkinson patients with concurrent accelerometry-fMRI. Frame-by-frame fluctuations in accelerometry-derived tremor power, measured per patient, were used to localize tremor-related brain activations and deactivations at a group level. Amplitude-related coupling between tremor-positive and tremor-negative brain networks was examined further using physio-physiological interaction analyses. Lastly, whole-brain multiple regression analyses were used to test the association between tremor-related activity and clinically assessed tremor severity. Tremor-related activations were found in a lateralized network consisting of contralateral (to tremor) sensorimotor cortex, ventrolateral thalamus, posterior putamen, and cerebellum (ipsilateral lobule V, vermis, and bilateral Crus I/II); activity in this network correlated positively with tremor severity. Tremor-related deactivations encompassed a bilateral "anti-tremor" network, including dorsal premotor cortex, pre-supplementary motor area, somatosensory cortex, posterior parietal cortex, and insula; activity in this network correlated negatively with tremor severity. During low versus high tremor episodes, functional connectivity between the anti-tremor and tremor-positive networks increased. This suggests that the anti-tremor network makes a relevant contribution to modulating tremor amplitude by connecting to the cerebello-thalamo-cortical circuit in a tremor-dependent manner. We conclude that spontaneous fluctuations in Parkinson's tremor amplitude are associated with interactions between a tremor-producing cerebello-thalamo-cortical circuit and a novel antagonistic anti-tremor network. Future intervention studies may investigate whether strengthening of the anti-tremor network leads to a reduction in tremor, as a potential basis for new therapies.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with limited therapeutic options. Riluzole remains the only widely available disease-modifying treatment for ALS, yet its survival benefit is modest and likely to vary substantially between patients. Cytochrome P450 2D6 (CYP2D6), is a highly polymorphic enzyme that contributes to interindividual variability in the metabolism of many drugs. CYP2D6 is also expressed in the brain, and experimental and translational studies indicate that brain CYP2D activity can influence local metabolism of neuroactive compounds. Accordingly, CYP2D6 poor function variants have been examined as susceptibility modifiers in the development of other neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease, with heterogenous evidence; however, the role of CYP2D6 in ALS has not been established.

Neuroinflammation and astrocyte dysfunction are associated with schizophrenia. To elucidate the molecular mechanisms involved, we generated induced pluripotent stem cells from 14 schizophrenia patients and 14 healthy controls and differentiated them to human cortical spheroids. Transcriptional profiling revealed overexpression of cilia-related genes in schizophrenia spheroids which was mainly driven by astrocytes. We identified baseline abnormal astrocyte distribution in schizophrenia and show that it is triggered by the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF), primarily secreted by neurons. While treatment with the MIF antagonist ISO-1 attenuated abnormal astrocyte distribution in schizophrenia spheroids and reduced pro-inflammatory cytokines secretion, MIF gene knockout with CRISPR-Cas9 exacerbated astrocyte and cytokines dysregulation in schizophrenia cultures, suggesting that both extremes of elevated and absent MIF lead to impaired astrocyte distribution and that a minimum expression of MIF is required for optimal cell function. Taken together, our results point to a MIF-induced regulation of cortical astroglia in schizophrenia and highlight MIF antagonists as potential novel treatment strategies.

Parkinson's disease is characterized by the accumulation and propagation of α-synuclein pathology in the central nervous system, yet the contribution of peripheral α-synuclein sources remains unclear. Here, we identify erythrocytes as an important reservoir of α-synuclein and demonstrate that bone marrow-derived erythrocytic α-synuclein likely contributes to brain pathology and Parkinson's disease-related neurodegeneration. Using human tissues and mouse models, we show that erythrocytes harbour abundant α-synuclein species. Bone marrow transplantation revealed widespread distribution of bone marrow-derived α-synuclein in peripheral organs, with detectable but substantially lower levels in the brain. Within the central nervous system, bone marrow-derived α-synuclein preferentially accumulated in resident microglia, as confirmed by immunophenotyping and single-nucleus RNA sequencing, and was associated with microglial activation. Furthermore, erythrocyte-derived extracellular vesicles carrying α-synuclein can be readily taken up by microglia in vivo. Functionally, elevated levels of bone marrow-derived α-synuclein in the mouse brain resulted in dopaminergic dysfunction with a mild neurodegenerative phenotype under baseline conditions. Importantly, blood-brain barrier integrity critically regulated peripheral α-synuclein entry into the central nervous system. Disruption of the blood-brain barrier by endotoxin administration, mannitol treatment or focused ultrasound markedly increased the entry of peripheral α-synuclein into the brain, aggravating neurodegeneration and behavioural deficits. Collectively, these findings identify bone marrow-derived erythrocytic α-synuclein as a systemic contributor to the pathogenesis of Parkinson's disease and highlight blood-brain barrier integrity as a key permissive regulator of peripheral-to-central α-synuclein transmission.

Infection-associated chronic illnesses (IACIs) encompass a spectrum of poorly understood syndromes often marked by significant neurologic and multisystem symptoms following an infectious event. This review focuses on several diseases representative of the IACI spectrum. These are post-treatment Lyme disease syndrome (PTLDS), long COVID, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and multiple sclerosis (MS). Their clinical and biological complexity, combined with a lack of clear diagnostic criteria and objective available laboratory biomarkers, makes them difficult to distinguish from conditions with overlapping features. This presents challenges for research studies, as well as diagnosis and clinical management. This diagnostic ambiguity, coupled with heterogeneous patient presentations, has led to challenges in research, including misclassification of study participants and inconsistent or irreproducible findings. Some PTLDS research exemplifies these issues, which also extend to other IACIs. To advance the field, we highlight key methodological refinements and approaches for studying IACIs, including rigorous participant selection, standardized sample collection protocols, and the use of appropriate control groups, including those with microbiologic proof of the initial infection when known and technologically feasible. We also address broader influences on research quality, such as stigma, historical neglect, and the urgency to find treatments, which have contributed to the proliferation of poorly controlled studies and questionable practices. Drawing lessons from past challenges, we propose a path forward grounded in fit-for-purpose methodological rigour to improve scientific understanding and support evidence-based therapeutic development for IACIs.

Regulatory T cells (Tregs) are essential for preventing autoimmunity. They depend upon interleukin-2 (IL-2) for optimal function and due to high expression of the CD25 subunit of the IL-2 receptor, are 10-fold more sensitive to IL-2 than effector T cells (Teffs). Consequently low-dose IL-2 can be used to preferentially expand Tregs, a therapeutic strategy which has shown promise in a number of autoimmune and inflammatory conditions - including graft-versus-host disease and lupus, where IL-2 driven expansion correlates with improvements in some clinical markers of disease activity. Autoimmunity is a frequent delayed complication of treatment with the lymphocyte-depleting drug alemtuzumab in relapsing-remitting multiple sclerosis (RRMS). Here, using in vitro assays, a pre-clinical mouse model, and an experimental medicine study, we investigated whether low-dose IL-2 could selectively expand Tregs in alemtuzumab-treated RRMS patients. Six months after alemtuzumab treatment, the frequency of patient-derived naïve CD4+ Teffs expressing high-affinity IL-2 receptors increased from 30.11+/-5.09% to 72.88 +/-5.57%, and the density of receptors per cell increased, rendering them six times more sensitive to IL-2 in vitro, at concentrations that typically favour Tregs. Using a human CD52-expressing transgenic mouse model of alemtuzumab treatment, we found that IL-2 was still able to preferentially expand Tregs, but only when administered at a later time point, corresponding to more than 6 months post-treatment in patients. Guided by these findings, we evaluated low-dose IL-2 for Treg expansion in a prospective open-label mechanistic study of RRMS patients who had received alemtuzumab more than 6 months previously. IL-2, at a dose and frequency similar to that previously shown to expand Tregs in autoimmune diabetes (0.3 x 106 IU/m2 twice a week), was well tolerated and safe, but unexpectedly failed to expand Tregs. We discuss the potential reasons underlying this lack of response, ruling out sIL2RA-related neutralisation, and instead considering ceiling effects on Treg proliferation, Treg exhaustion and intrinsic Treg dysfunction in MS as possible contributors. Together, our findings demonstrate that low-dose IL-2 alone is not an effective strategy for promoting Treg expansion post-alemtuzumab, and is therefore not a viable approach by itself for preventing post-treatment autoimmune complications.